Tyrosine kinase inhibitors relax pulmonary arteries in human and murine precision-cut lung slices

Abstract

Background Tyrosine kinase inhibitors (TKIs) inhibit the platelet derived growth factor receptor (PDGFR) and gain increasing significance in the therapy of proliferative diseases, e.g. pulmonary arterial hypertension (PAH). Moreover, TKIs relax pulmonary vessels of rats and guinea pigs. So far, it is unknown, whether TKIs exert relaxation in human and murine pulmonary vessels. Thus, we studied the effects of TKIs and the PDGFR-agonist PDGF-BB in precision-cut lung slices (PCLS) from both species. Methods The vascular effects of imatinib (mice/human) or nilotinib (human) were studied in Endothelin-1 (ET-1) pre-constricted pulmonary arteries (PAs) or veins (PVs) by videomicroscopy. Baseline initial vessel area (IVA) was defined as 100%. With regard to TKI-induced relaxation, K + -channel activation was studied in human PAs (PCLS) and imatinib/nilotinib-related changes of cAMP and cGMP were analysed in human PAs/PVs (ELISA). Finally, the contractile potency of PDGF-BB was explored in PCLS (mice/human). Results Murine PCLS: Imatinib (10 μM) relaxed ET-1-pre-constricted PAs to 167% of IVA. Vice versa, 100 nM PDGF-BB contracted PAs to 60% of IVA and pre-treatment with imatinib or amlodipine prevented PDGF-BB-induced contraction. Murine PVs reacted only slightly to imatinib or PDGF-BB. Human PCLS: 100 μM imatinib or nilotinib relaxed ET-1-pre-constricted PAs to 166% or 145% of IVA, respectively, due to the activation of K ATP -, BK Ca 2+ - or K v -channels. In PVs, imatinib exerted only slight relaxation and nilotinib had no effect. Imatinib and nilotinib increased cAMP in human PAs, but not in PVs. In addition, PDGF-BB contracted human PAs/PVs, which was prevented by imatinib. Conclusions TKIs relax pre-constricted PAs/PVs from both, mice and humans. In human PAs, the activation of K + -channels and the generation of cAMP are relevant for TKI-induced relaxation. Vice versa, PDGF-BB contracts PAs/PVs (human/mice) due to PDGFR. In murine PAs, PDGF-BB-induced contraction depends on intracellular calcium. So, PDGFR regulates the tone of PAs/PVs. Since TKIs combine relaxant and antiproliferative effects, they may be promising in therapy of PAH.

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays

“…We developed a new protocol that allowed us to efficiently cut mouse spleens in intact slices and to maintain these alive and responsive for at least 48 h, making them suitable for functional assays. The protocol, that is a modification of protocols developed to obtain precision-cut slices of mouse brain, liver and lung (9)(10)(11), is based on the sequential following steps: (1) spleen inclusion into agarose blocks; (2) precision-cut using a vibrating microtome; and (3) 48-h culture of spleen slices. The protocol developed for the preparation of organotypic cultures of mouse spleens has turned out to be a valuable tool to (i) prepare spleen slices with a sufficient degree of tissue integrity; and (ii) maintain this complex tissue in culture for days, in order to be used for functional assays.…”

Levosimendan reduces segmental pulmonary vascular resistance in isolated perfused rat lungs and relaxes human pulmonary vessels

“…The meaning of the studied species is supported by several studies, e.g. pulmonary vessels from humans PLOS ONE and guinea pigs varied in their reaction to imatinib [23,24], PDGF-BB [24,45] or milrinone [31]. Finally, to obtain valuable information concerning the vasorelaxant effects of several vasodilators in the human pulmonary vascular bed, human PAs and PVs should be studied.…”

“…So far, it is unexplored, whether levosimendan also exerts relaxation in a more peripheral part of the lung. Third, remarkable differences exist between various species [23][24][25]; hence we do not know if levosimendan-induced relaxation is also relevant for human PAs and PVs.…”

Repurposing of medications for pulmonary arterial hypertension

“…PCLS is a technique that permits culturing of human lung sections. 65 In PAH research, PCLS were used to show the tyrosine kinase inhibitors, imatinib and nilotinib, promote vasodilation in preconstricted vessels, 66 and that substance P promotes pulmonary vascular remodelling. 67 However, use of PCLS for drug screening purposes in PAH has not been implemented yet.…”